1. Field of the Invention
The present invention is related to systems for detecting target ions in a sample, and more specifically, to ion-detecting microspheres and methods of use thereof in clinical laboratory instrumentation such as flow cytometry for sample analysis.
2. Description of the Prior Art
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
The analysis of complex biological fluids, such as whole blood, serum, and urine, is of paramount importance in clinical chemistry. Electrolytes such as sodium ions and potassium ions are routinely assessed using carrier-based ion-selective electrodes (ISEs) (1-2). With more than one billion ISE measurements being performed annually world wide in clinical laboratories, this class of chemical sensors plays a crucial role in laboratory diagnostics. Trends in analytical chemistry continue to move toward the development of miniaturized systems, and there is a great interest in streamlining all available assays into one common method. One approach towards achieving this goal is the use of optical readout methodologies.
Several approaches have been developed for ion analyses that use fluorescence transduction (3-17). This is primarily due to the high signal-to-noise ratio afforded by this detection method, making it an attractive choice for creating sensors of reduced size. Most ion-selective optical sensors use the same carriers previously developed for use in ISEs, and they obey bulk extraction principles consistent with traditional optode theory (2).
Typically, an optode membrane is composed of a plasticized poly-(vinyl chloride) (PVC) matrix, an ionophore that selectively binds the primary ion, ionic sites that facilitate mass transfer of ions from the aqueous sample to a hydrophobic sensing phase, and a hydrogen ion-selective fluorescent chromoionophore (fluoroionophore), which is responsible for signal transduction. Other approaches for ion determinations utilized particle-based technologies. Lubbers et al. have reported optical nanoprobes for measuring the pH and pO2 of physiological structures (10). More recently, Kopelman et al. reported both acryl amide and PVC-type nanometer-sized sensing spheres that have proven to be quite useful in interrogating intracellular environments (3, 4, 5, 19). Bakker and group have prepared micrometer-sized particles by various methods, including heterogeneous polymerization (13), solvent casting (17), and very recently, with the use of a sonic stream particle casting apparatus (16). Spatial and spectral characterization of the particles was performed by fluorescence microscopy/spectroscopy. However, the use of this technique has not been described for high throughput screening applications prior to the present invention.
In an attempt to increase the throughput of ion determinations, Kim et al. have described a 96-well plate-format absorbance-based optode that requires micro volume samples and that could be read using existing clinical laboratory instrumentation (20). An even more promising technique, however, that offers rapid, high-throughput analyses with multiplexing capabilities is flow cytometry. Microspheres have been used for flow cytometric applications for more than 25 years (21), and they are commonly used for multiplexed analyses. It has been demonstrated that as many as 64 different analytes can be screened simultaneously using microsphere-based technologies (20-24). With large surface area available for attaching numerous molecular recognition chemistries (6) and a core that can be impregnated with encoding dyes (25), microspheres have played an important role in the development of suspension array technologies (26). Numerous biologically relevant analytes have been detected using microsphere-based cytometry (7, 8, 11, 12, 24, 27-29). Electrolytes, however, are a class of analytes that have never been assessed with this technique.
Lower detection limits, smaller sample volumes, faster response times and high selectivity are among the many requirements that must be met in the trace level analysis of complex samples. Towards this end, optode films based on neutral ionophores have proven to be a highly promising technology for the analysis of heavy metal ions. Over the past decade, an increasing number of cation-exchange based systems including those for Pb2+, Cu2+, Hg2+, Ag+, UO22+, have been reported (30-34).
For the analysis of lead ions, optode systems that incorporate highly selective ionophores have been used. Those that may contain sulfur coordinating functionalities, like a calixarene bearing a —SH pending group or a di-thioamide derivative, have demonstrated increased success in polymer membranes of ISEs where they have been shown to exhibit detection limits that extend even to picomolar levels with electrodes that are carefully tailored (35, 36). In optode systems, selective ionophores incorporated with a lipophilic chromoionophore and required anionic sites have been examined with view to their use in environmental monitoring (30). As for all conventional cation-exchange based systems, lead optodes follow predicted theory as given by the associated equilibrium of transfer of the lead analyte species and hydrogen ions into the plasticized PVC optode phase. In particular, the lead complexing agent 3,6-dioxaoctanedithioamide derivative, that is, ETH 5435, in conjunction with the absorption changing properties of chromoionophore ETH 5418, was found to exhibit excellent selectivity against all relevant alkaline and alkaline-earth metal ions, thus allowing lead measurable concentrations to extend to the sub-nanomolar range.
Antico et al. (37) have reported on optode films incorporating the ionophore ETH 5493, which is a mono-thio oxodiamide derivative of ETH 5435. While the use of such an ionophore offers less good selectivity with respect to the alkaline-earth metal ions Ca2+ and Mg2+ than the di-thioamide ligand, no irreversible sensing film poisoning upon exposure to Ag+ or Hg+ ions does occur.
These papers on ionophore-based optodes have shown that the selectivity and detection limits are sufficient to reach sub-nanomolar detection limits. However, a significant drawback is the high sensing volume of the optode film, which requires typically on the order of 10 μmoles of ions to be extracted from the sample in order to achieve the desired optical response. Consequently, massive volumes of sample (many liters) and very long response times (many hours) in a continuous flowing system have so far been required to accurately measure low levels of heavy metals in aqueous samples. A drastic miniaturization of the sensing element should be able to alleviate this important problem.